This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic, 06-CA-117: Cancer Development, Pathology, and Pathological Progression. B cell chronic lymphocytic leukemia (CLL) is the most common leukemia affecting adults in Western countries and is recognized by the accumulation of a clonal population of B lineage cells with a surface phenotype distinguishing them from most mature B cells. Although the disorder has been know for over thirty years, surprisingly, its etiology remains unclear and standard treatments, established twenty years ago, entail significant side effects and do not affect a cure, but rather delay progression for a time. In the past ten years it has become clear that CLL can be subdivided into two categories based on extent of mutation of the B cell antigen receptor (BCR): those with significant mutation (>2%) show slower progression and generally a more benign course, whereas those with little mutation have a much worse prognosis and faster progression. Sequence analysis of heavy chain variable regions (VHs) of the BCR has revealed intriguing findings indicating significant VH gene usage biases, and the recurrent usage of similar or even identical CDR3 segments, suggesting a role for selection by common antigens in development of either CLLs or their precursors. There is also clear evidence that CLL BCRs often encode autoreactivity and such self-reactivity may play a key role in the course of disease. Furthermore, recent studies have described the presence of CLL-like monoclonal expansions in relatives of CLL patients, but also more generally in adults over the age of 60. As CLL is a disease whose incidence increase with age, rarely found in young individuals, it is tempting to speculate that clonal B cells often accumulate with age, but only infrequently progress to the CLL stage. Understanding the factors that generate these monoclonal B-cell lymphocytoses (MBLs) and determining how they progress to CLL could lead to breakthroughs in the treatment of CLL. Our interest over the past twenty years has focused on development and characterization of one kind of autoreactive B-cell in the mouse that may model the cell type with a propensity to become MBL and eventually, CLL. These cells, initially identified by expression of CD5 (previously called Ly-1 in mouse), show distinctive localization and functions, importantly being responsible for much of the natural autoantibody production in mice. Some years later, we (and others) showed that production of autoantibody rheumatoid factor was enriched in human CD5+ B-cells. We now think the time is ripe to ask whether generation of the striking biases seen in V genes of BCRs from both mouse and human CD5+ B-cells and CD5+ B-cell leukemias may involve, at least in part, an as-yet un-appreciated mechanism that sculpts the repertoire of natural autoreactive B cells. That is, at the pre-B stage of development, there is strong selection based on assembly of a pre-BCR, and this _-selection checkpoint may be important in understanding the development of distinctive heavy chains with an autoreactive bias. We have developed assays using mouse cells for measuring the capacity of Ig heavy chains to assemble into a pre-BCR and correlated this with the induction of proliferation. We now propose to extend this work, examining heavy chains used in a series of late-developing CLL-like mouse leukemias, but importantly also establishing comparable assays with human cells. This will allow us to assess of pre-BCR assembly and signaling in the human system, and then examine heavy chains from CLL and MBL, to ask whether the distinctive heavy chain biases seen in these cells arise much earlier in development, potentially providing important new insights into the growth and eventual transformation of such cells. Our proposed work will establish the groundwork needed for further study of the role of BCR in development of CLL precursors, eventually uncovering new treatments. Finally, an important aspect of this project is that it will apply our extensive experience and expertise in mouse B cell development, selection, and subsets in a focused study of a potential etiology of a human leukemia, with the potential of developing better, more targeted, therapies. PUBLIC HEALTH RELEVANCE: This project seeks to understand whether chronic lymophocytic leukemias (CLL), the most common leukemia in adults, arises from a distinct set of natural autoreactive B cells, by testing a property of the antibody present on such cells that distinguishes them from other types of B cells. This will be done using a mouse model for CLL, and also by obtaining patient leukemic cells and directly testing their antibodies. The knowledge generated in this study should provide insights into the origins of CLL and lead to the development of new, more targeted, therapies, improving patient quality of life and potentially even providing a cure for this disease.